Agglutination reactions involve the crosslinking of carrier particles coated with antigen (antibody) by the appropriate antibody (antigen). Agglutination reactions can also involve crosslinking of carrier particles by binding molecules other than antigens and antibodies (e.g. receptor and ligands). Agglutination reactions provide an important nonisotopic method for immunoassay of high sensitivity and specificity. The sensitivity and specificity of agglutination reactions have been substantially enhanced by application of techniques which are capable of detecting the cross-linking process at an earlier stage of the agglutination process. See, e.g., Cohen et al. U.S. Pat. No. 4,080,264 (quasi-elastic light scattering) and Cannell et al. U.S. Pat. No. 4,174,952 (light scattering intensity anistropy). By replacing the visual detection of macroscopic agglutinates containing hundreds of thousands of carrier particles as the reaction end point with an optical technique of measuring the extent of reaction--the agglutination reaction is transformed from a qualitative, and fairly sensitive immunoassay, to a quantitative and more sensitive immunoassay method.
Both the quasi-elastic light scattering spectroscopy and intensity anisotropy methods involve measurements made on bulk solution and reflect essentially different moments of the cluster size distribution of the cross-linked carrier particles. It has been estimated that the quasi-elastic light scattering spectroscopy can reliably detect agglutination when roughly 25% or more of the carrier particles are agglutinated, the intensity anisotropy method has been estimated to when as little as 5% of the carrier particles are agglutinated.
Although quasi-elastic light scattering and optical anisotropy methods of detecting agglutination provide a fairly sensitive immunoasssy, these methods cannot discriminate between light scattering from specific size clusters of carrier particles and artifacts such as dust particles or cell fragments which can cause markedly erroneous results when bulk detection techniques are used.
Another potential particle counting technique for immunoassay might be based on resistive-pulse method (or impedance). See e.g., von Schulthess, G. K., et al. Macromolecules 13:939 (1980); von Schulthess, G. K., et al. Macromolecules 16:434 (1983). The electrical, or resistive-pulse technique consists of suspending the particles in an electrolytic solvent so that as each particle enters the pore it displaces a portion of solvent resulting in a change in the pore's electrical conductivity. Since the change in conductivity i proportional to the volume of the particle, the size distribution of the sample is accumulated by sending thousands of particles through the pore. Because the measurement and subsequent signal processing is rapid, statistically meaningful distributions can be obtained in minutes.
However, resistive pulse analysis is lacking in several respects.
(1) For one, the resolution of the resistive pulse method with regard to its ability to distinguish between sizes is limited. This restricts the sensitivity of such a method because a count allocated to dimers might in fact be a monomer or vice versa.
(2) The resistive-pulse technique cannot differentiate coincidence of two monomers within the pore and the presence of one dimer because they both displace the same volume of electrolyte.
(3) The pores of the detection cell are subject to frequent plugging limiting its practical application.
Additionally, a resistive pulse analyzer requires that the particle be suspended in a high ionic strength solution in order to reduce electrical noise. Such high ionic strength may not be compatible with the formation of specific antigen antibody bonds and may nonspecifically promote, or inhibit, the clustering of the carrier particles.
The cumulative effect of the above makes the resistive-pulse analyzer an impractical means of achieving high sensitivity immunoassay based on agglutination reaction.